JPS63119428A - Adoptive immunotherapy for treating aids - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [1] Field of the Invention The present invention relates to the use of adoptive immunotherapy in the treatment of patients infected with the AIDS virus.
in vitro activation of T cells that mediate the immune response against the AIDS virus,
including expansion of these T cells,
It is then inoculated into a patient, injecting into the patient a cell-mediated immune response that is specifically directed against AIDS virus infected cells. The present invention provides for the isolation, in vitro activation, demonstration of immune specificity, expansion and administration of activated lymphocytes, which can be used to mediate in vivo T cell immune responses against AIDS virus antigens.

The method of the invention involves isolation of lymphocytes from a patient or a histocompatible donor whose immune system has been exposed to AIDS virus antigen(s) and in vitro activation of the lymphocytes by exposure to AIDS virus-related epitopes. . Expansion of T lymphocytes reactive to AIDS virus antigens and inoculation of T cells into patients suffering from AIDS virus-related disorders may provide a useful form of immunotherapy.

(2) Background of the Invention (2,1) AIDS virus acquired immunodeficiency syndrome (AIDS) is a disease identified by severe immunodeficiency caused primarily by impairment of the patient's cell-mediated immune response [Gottlieb ,
M, et al., 1981, Niney English Journal of Medicine (N, [Engl.

JoMed, ), 3 (15: 1425; Masur, J., et al., 1981, N., En.
gl, J9Med,), 3 (15:1431). Two clinical manifestations of the disease: (a) chronic lymphadenopathy, leukopenia and a normal peripheral helper to suppressor T lymphocyte ratio (○KT4) that shifts from 2 to 0.1 or less as the disease progresses; : 0KT8), a prodromal phase known as lymphadenopathy syndrome confirmed by a quantitative decrease in peripheral blood helper cells (OKT4 cells) resulting in a reversal of 0KT8), and (b) a decrease in OKT4 cells and normal 0KT4
:0KT8 ratio reversal, absolute lymphopenia, and mainly Pneumocystis carinii
An immunodeficiency condition known as AIDS, confirmed by recurrent opportunistic infections with S. carnii), is recognized, and this latter stage is often associated with eventual death.

Certain subpopulations of patients have a high incidence of lymphoma and Kaboji's sarcoma. There is currently no cure for the disease.

Epidemiological data as well as information related to the type of patients who acquired the disease suggested that infectious agents transmitted by intimate contact may be the cause of the disease. Three groups subsequently provided solid evidence that the causative agent of AIDS is a retrovirus with tropism for T helper lymphocytes. These groups are (a) R0C9Gallo and collaborators from the National Institute of Health;
We were able to isolate a cytopathic retrovirus (HTLVII) from AIDS and pre-AIDS patients [Gallo, RlC, et al., 1996].
84, Science, 224:
5 (11): Popovic (M,)
et al., 1984, Science
, 224: 497: ] o They also detected antibodies against HTLVI in the serum of patients with AIDS.

Montagnier, L. and co-workers isolated a T-lymphotropic retrovirus (LAV) from a patient at the Pasteur Institute who presented with cervical lymphadenopathy and was at risk of needing treatment [Barre et al.].
Barre-Sinoussi, F. et al., 1983, Science, 22
0:863]. This group was also able to show antibodies against LAV in the serum of AIDS patients [Kalysansraman, V.
S.) et al., 1984, Science.
, 225:321]. Furthermore, they were able to isolate LAV from the lymphocytes of patients who developed AIDS after receiving blood from a donor who developed AIDS [Feorino, PoM, et al., 1984;
Science, 225: 69
].

(c) Levy (J, A. , ), 19
84, Science, 225:8
40].

It is.

Although all three viruses were isolated independently, they all probably belong to the same retroviral subgroup (Levy et al., 1984, Science, 225:840).
Here they are collectively denoted as LAV/HTLVI[I.

The general structure of toreroviruses is that of a ribonucleoprotein core surrounded by a lipid-containing envelope that the virus acquires during the process of cell budding. The glycoprotein encoded by the virus is embedded within the envelope and protrudes outward. This determines the host range of the virus and reacts with specific receptors on the surface of susceptible cells. Neutralizing antibodies appear to bind to envelope glycoproteins and block their interaction with receptors on the surface of cells.
“The Molecular Biology of Tumor Viruses”
arBiology of Tumor Viruse
s) J, John Tooze
ed., 1973, Cold Spring* Bar Bar
* Laboratory (cold Spring Harb)
orLaboratory), pp. 534-535;
rRNA Tumor Virus
ses) J, Weiss et al. (R.

Weiss, N., Te1ch, H. Varmus
and J, Coff1n) m.

1982, Cold Spring Harbor Laboratory
ratory), pp. 226-227 and 236-
237 pages].

There is evidence that the T4 antigen, which is present on a subset of T lymphocytes in certain cases of LAV/HTLVIII, is the receptor or part of the receptor for the virus [Dalgleish, (Dalgleish, 1993).

A, G, et al., 1984, Naech, ? (Natur
e), 312 Near 63; Kula 7? 7 (Klatz
Mann, D., et al., 1984, Nature (N.
ature), 312 near 67).

The RNA genome of LAV/HTLVIII contains the gag gene, which encodes the internal structural protein (core protein) of the virus, the pal gene, which encodes the viral group-specific antigen virion-associated reverse transcriptase, and the viral glycoprotein. Define the env gene. Other regions, such as sor and 3'-orf, represent regions of the genome that contain open reading frames; the functions of these regions are currently unknown.

(2,2) Cell-Mediated Immunity to Retroviral Antigens Little is known regarding the antigenic specificity of T cells to retroviruses. But Friend and Mooney
? It has been reported that the envelope glycoprotein of murine leukemia virus (MuLV), gp70, is recognized by MuLV-immunized mouse T cells [Matis et al.
l, ^, 〉 et al., 1985, Journal of Immunology (J, Immunol, ), 135 Near (
13-7 (15; Flyer, D, C,
) et al., 1983, Nature, 3(
15: 815-818).

Plaque (Plata) [Abstract, communication (commun 1 caton), 1
61:527k.

International Conference on AIDS
ConferenceOn Al(13), 1986
June 23-25, 2006, Barry, France] showed that mice can generate cytotoxic T cells against envelope or gag antigens expressed on transfected MuLV-induced tumors. T directed against cells infected with human T lymphotropic virus infection (HTLV-I)
Lymphocytes were shown [Mitsuya, Hl
) et al., 1983, Journal of Experimental Medicine (J, EXIT9Med,), 1
58:994-999], but no recognized antigen was reported.

(2,3) In addition to in vitro activation of virus-directed T cells, T cell-mediated immunity plays an important role in resistance to or recovery from diseases caused by many and various enveloped viruses. It has been shown. We show that T lymphocytes capable of specifically recognizing virus-infected cells can be generated in vitro by stimulating lymphocytes from virus-infected or immunized animals with viruses, viral proteins, or viral peptides. There has been some research. An example in mice is the influenza virus [2weerink.

HoJ, ) et al., 1980, Nature
e), 267:3'51), herpes simplex virus (H
8v) [Lawman, M, J, P,
'et al., 1980, Infection and Immunity (Infec.

Immun, ), 30:451], or murine leukemia/sarcoma virus [Fernandez C.
andez-Cruz, ε, ) et al., 1979, Journal of Immunology (J, Immunol, ),
123:1772:], which specifically recognizes infected cells. Such sensitized T cells have cytotoxic T lymphocytes (or cytotoxic T lymphocytes) as measured by the ability of the T cells to proliferate and produce lymphokines in response to stimulation by a virus or viral antigen.
(denoted as CTL) and/or T helper cell activity. Evidence that in vitro activated T cells can be useful for immunotherapy when inoculated into experimental animals is that virus-activated murine T cells or Till cell clones can be used for influenza [Lukacher, Hachi, et al. , 1934, Journal of Experimental Medicine (J
.

EXll, λ1ed), 160:814), lymphocytic choriomyelopancreatitis virus [Byrne et al.
, R, andoldstone, M, B, A,)
, 1984, Journal of Pyrology (J, Vi
rol), 51:682), herpes simplex virus (H
SV) Larsen, H, L, et al., 1984, Journal of Biology (J, V
irol, ), 50: 56; Sethi
, K., K.) et al., 1983, Journal of
Pyrology (J, Virol, ), 64: 44
3) or murine leukemia virus [chee ver
ver.

M, ^,) et al., 1986, Journal of Experimental Medicine (J, Mi xp, , Me
d, ), 163:11 (11)) based on the finding that resistance to relapse or death caused by viruses can be adoptively transferred. In addition, interleukin 2 (IL
-2) murine lymphocytes non-specifically activated and designated as lymphokine-activated killer (LAK) cells have been shown to be immunotherapeutically useful in eliminating metastases from various murine tumors [ Rosenb
erg, S. A.), 1985, J.

Natl, Cancer Dist;, 75:
595: Mazumder et al.
, and Rosenberg, l 934, Journal of Experimental Medicine: /(J,
Exp, Med, ), 159:225:].

Peripheral blood lymphocytes (PBL) with viruses or viral antigens
) It was also possible to activate human T helper and/or cytotoxic T cells by stimulating them. For example, HSV (Sethi, K, K
) et al., 1980, Nature, 2
70:529; Yasukawa et al.

M. and 2arling, J.), 1984
, Journal of Immunology
ol, ), 133:422] or influenza virus [Biddison, B.W.
) et al., 1981, Journal of Immunol, 122:660; McMichael et al.
dAskonas, B. 8), 1979, European Journal of Immunology (Miur, J.
Human) T cells that specifically recognize [mmunol), 5nia (15) were generated in vitro. Purified HSV in the H3V system.

H3Va proteins cloned and expressed in mammals, and individual H5V peptides with H5V specificity)
It has been reported that Zaring (Za) activates cells.
Rling, J. M., et al., 1986, Journal of Immunology (J. Immunol, ).
136:4669; DeFritas (DeFritas,
E.

C, et al., 1985, Proceedings of the National Academy of Sciences (Pro
c, Natl, Acad, Sci,)
[ISA, 82:3425:].

Although the use of in vitro sensitized uvirus-activated T cells was not reported, human LAK cells have recently been used in cancer patients.
It has been reported that inoculation with L-2 can cause regression of various tumors [Rosenberg (R.
Osenberg, S., et al., 1985, New England Journal of Medicine (
N, Eng, J, Med, ), 313:1485)
. However, LAK cells can also produce similar side effects associated with grafts to host disease. These side effects may be due in part to the fact that normal lymphocytes, in addition to cancer cells,
This can be explained by the recent observation that AK cells can be killed by [Sondel, P.
oM, ) et al., 1985, Science
e), 228:1185].

(2, 3, 1) Research with recombinant vaccinia virus A recombinant vaccinia virus expressing antigens of a foreign virus was found to induce resistance to challenge by a foreign virus in experimental animals.

Examples include H3V glycoprotein [cremer.

K, J.) et al., 1985, Scien
ce), 228:1985L rabies virus surface antigen [
Blancou, J. et al., 19
86, Nature, 322:373)
, and influenza virus nucleoprotein [Smith.

G, L, et al., Proceedings of the National Academy of Sciences) USA.

80 near 155; Yewdell, J.
W. et al., 1985, Proceedings of the National Academy of Science (Proc.
, Natl.

Acad, Sci.), USA, 82:17
85) is included. A recombinant vaccinia virus expressing influenza virus nucleoprotein was reported to induce influenza virus-specific T cell-mediated immunity in immunized mice [Benn.
ink, J, R, et al., 1984, Nature (N
Furthermore, using target cells infected with recombinant vaccinia virus expressing influenza virus nucleoprotein, influenza virus nucleoprotein was HJed by cytotoxic Till cells from influenza seropositive blood donors. was shown [McMichael (1, lc! Jichael,
A, J.) et al., 1986, Journal of General Biology (J. Gen. Virol, ).

67:719:]. Similarly, we recently observed that human target cells infected with a recombinant vaccinia virus expressing the H3V glycoprotein were 171m infected with a human CTL clone specific for E(SV) [Zarring et al.
ling, J, M, et al., 1986, Journal of Pyrology (J, Virol, ), 59:50
6). We also used a recombinant vaccinia virus expressing the H3V glycoprotein to detect BP in humans infected with H8v.
specific for H3V-infected cells by stimulating L.
Ti [one cell clone could be generated [Zarling et al., Journal of Pyrology (J, Virol, supra)].

(3) Summary of the invention The present invention provides T +
A method using in vitro activation of Jumba spheres is directed. Lymphocytes of the invention include, but are not limited to, AIDS or AIDS-related complex (ARC) patients themselves and HLA-compatible blood donors who have been immunized by natural exposure with the AIDS virus or by inoculation with AIDS virus-related epitopes. It can be isolated from individuals whose immune systems have been exposed to viral antigens. The phosphorus/(
The bulbs were immunized within the test by exposure to peptides or proteins related to epitope(s) of the AIDS virus.
li11 is furious. These epitopes may be present in compositions containing recombinant vectors, e.g. viruses, or naturally occurring
It can be included in purified proteins or peptides, either recombinant or synthetic.

The present invention provides activated T lymphocytes prior to inoculation into patients;
and expansion of activated lymphocytes in cell culture to demonstrate reactivity to AIDS virus epitopes. Inoculation of patients with AIDS virus-reactive T cells may be accompanied by other therapeutic controls such as IL-2, interferon, anti-L
It may or may not involve AV/HTLVIII antibodies, antiviral drugs, bone marrow transplantation, etc.

Very recently, LAV/LAV by cytotoxic T cells (cTL) and lymphokine-activated killer (LAK) cells
Lysis of HTLVIII-infected phosphorus/-, 8 bulbs has been described [Koenig, S. et al.
1 on), 26: U3a, International Conference on AIDS 1 (
Inter-national conference
on A1[IS], June 23-25, 1986,
Barry, France]. Evidence was found for the presence of cytotoxicity against LAV/HTLVI infected targets in AIDS virus seropositive individuals. In addition, Kro force (Kro
wka) Haka [Abstract, Comi 3 Nikation (
communication) 3 Q:S3e,z
International Conference on Identification 1% (Inter'tiona
Conference on AIDS), 1986
June 23-25, 2016, Barrie, France] showed that some AIDS virus-seropositive donor peripheral blood lymphocytes responded to recombinant AIDS virus antigens by depositing and/or
Provided evidence that IL-2 release occurs.

An advantage of the use of AIDS virus-specific T cells for immunotherapy of AIDS virus infection according to the present invention is that these T cells
The cells will recognize virus-infected cells but not normal cells.

Recent advances in engineering for large-scale culture of mammalian cells, including the continuous growth of Ti follicles in IL-2 [Feder, J., and Tolbert, I.
Q, ), 1983, Scientific American (Sci, American), 248: 36
; Artocinilla (Artschuller, G.
L., et al., 1986, Biotech and Bioengineering (Biotech, and
Bioengineering, 28:646) enables the study of adoptive immunotherapy to treat individuals infected with the AIDS virus.

In a particular embodiment of the invention, lymphocytes isolated from macaques and chimpanzees immunized with recombinant viruses expressing AIDS virus envelope determinants, and human lymphocytes obtained from patients seropositive for the AIDS virus. Lymphocytes were exposed in vitro to AIDS virus envelope epitopes and/or recombinant vaccinia virus expressing AIDS virus envelope epitopes to induce proliferation. Stimulated macaque lymphocytes were shown to be T cells that produced IL-2 after stimulation. Additionally, helper or cytotoxic T cells have been generated in chimpanzees.

(4) DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method for immunotherapeutic treatment of patients infected with the AIDS virus, which involves in vitro activation of lymphocytes with AIDS virus-related epitope(s) prior to inoculation of the patient.

Activated T cells are expanded in cell culture and inoculated into patients infected with the AIDS virus, either alone or in combination with other therapeutic regimens, as a form of adoptive immunotherapy. Peripheral blood lymphocytes (PBL) can specifically recognize AIDS virus-infected cells and contribute to the elimination of the virus or virus-infected cells, but do not eliminate normal cells. A method of activation is described.

Such activated PBLs can extend their effects by direct killing of virus-infected cells or by inhibiting the production of infectious virus and/or by directing other cells to their role in acting on virus-infected cells. It can be mediated by the production of lymphokines that can be activated.

The method of the invention includes, by way of illustration only, the following steps: (a) isolation, in vitro activation, and expansion of T lymphocytes; (c) demonstration of induction of specific cell-mediated immunity; and (c) activation of T lymphocytes. Inoculation of T cells into a patient.

For clarity of discussion, the method is primarily discussed with respect to the use of recombinant vaccinia virus expressing the AIDS virus envelope glycoprotein. However, the same method can be applied to different AIDS virus-related epitopes, such as gag.
Use of other recombinant viruses expressing proteins, or produced by other recombinant DNA engineering or chemical synthesis, or viral particles, patient blood or blood? A similar method can be applied for the use of purified peptides or proteins such as blue.

(4,1)! Isolation, Activation, and Expansion of J Lymphocytes The lymphocytes used for in vitro activation are T Ly2 cells directed against AIDS virus epitopes. These T cells can be obtained by isolating peripheral blood lymphocytes (PBL) from individuals exposed to AIDS virus-associated epitopes and compatible with human leukocyte antigen(s) (HLA). .Such suitable individuals include the patient himself (
AIDS or an AIDS-related complex, ARC), or who have been exposed to AIDS virus-related epitopes by natural exposure to the AIDS virus or by inoculation with AIDS virus-related epitopes.

The present invention is directed to the use of peptides or proteins related to epitope(s) of the AIDS virus in in vitro stimulation of T lymphocytes.

These peptides or proteins include naturally occurring molecules that can be purified from AIDS virus particles, patient blood, serum, etc. by a variety of known techniques. for example,
LAV/HTLVIII envelope glycoprotein can be purified from disrupted LAV/HTLVIII by lentil lectin chromatography. Other possible purifications include other types of chromatography (e.g. ion exchange, affinity, sizing columns)
, centrifugation, differential solubility
protein purification), immunoprecipitation and preparative electrophoresis, or other standard techniques for protein purification.

Peptides or proteins used for stimulation can also be purified from recombinant viruses or other products of recombinant DNA technology. Peptides or proteins related to the AIDS virus epitope(s) for use in the invention can be produced in and isolated from host cell expression vector systems, such as in animals infected with the appropriate recombinant virus. or insect cell culture - including microorganisms such as bacteria transfected with recombinant plasmids, cosmids or phages; and yeast transformed with recombinant plasmids. Furthermore, recombinant microorganisms containing AIDS-related epitopes can themselves be used.

These microorganisms contain epitopes that, either in their normal state or after antigen processing by other cells such as macrophages or by treatments (e.g. denaturants, detergents, etc.) that denude AIDS-related epitopes, can be
will be exposed to the Umba sphere. Alternatively, peptides and proteins containing AIDS virus-related epitopes can be chemically synthesized and used to stimulate lymphocytes. Examples of AIDS virus-related epitopes that can be used in accordance with the present invention include co-pending U.S. Patent Application No. 779.90.
No. 9 (filed on September 25, 1985), No. 842.984
No. (filed March 27, 1986) and U.S. Application No. 562'4-026 filed September 9, 1986; Said application is hereby incorporated by reference.

Various known methods of enhancing the proliferation of stimulated T cells can be used and are within the scope of the present invention. For example, T
Antibodies or their derivative molecules that recognize Tp67 or Tp44 antigens on cells have been shown to increase the proliferation of activated T cells [Ledbetter,
J. A. et al., 1985, Journal of Immunology (J. Immunol.), 135:2.
3313, can be used to increase proliferation during in vitro activation. Interferon has been shown to increase the production of cytotoxic T cells [Zarlin et al.
g) et al., 1978, Immunol.
ogy), 121:2(11)2), can be used to increase the production of cytotoxic T cells against AIDS virus infected cells during in vitro activation.

The activated T cells can then be expanded in cell culture. This expansion can be accomplished by repeatedly stimulating T cells with AIDS virus-associated epitopes with or without IL-2, or by growth in medium containing IL-2 alone. Other methods of culturing T cells (eg, with other lymphokines, growth factors, or other bioactive molecules) are also within the scope of the invention.

Antibodies or antibody fragments that define T cell antigens such as Tp67 or Tp44 antigens that can support T cell growth and maintenance in vitro [Ledbetter (Le
dbetter.

J.A. et al., 1985, Journal of Immunology (J. Immunol.), 135:23:H]
Also within the scope of this invention is the use of

The general method of isolation, activation and expansion of PBL is then detailed, primarily with respect to the use of recombinant vaccinia virus containing AIDS virus enveloped shrimp cells. However, the present invention provides for the use of AIDS virus-related epitopes in a variety of forms, as described above, and variations thereof as well as modifications and adaptations of methods to accommodate various known manipulations are within the scope of the invention.

Approximately 2(11)- heparinized venous blood was withdrawn by venipuncture and PBLs were separated by Ficoll-Hypaque gradient centrifugation, approximately 1-5×10 depending on the donor's lymphocyte count.
' Get PBL.

PBLs were washed in phosphate at saline and incubated in RPM 11640 medium containing 10% pooled heat-inactivated normal human serum from AIDS-seronegative donors. : Approximately 2X
This medium, suspended at 10'/d, is designated as "complete medium". 750ml of cells! An ultraviolet (UV)-inactivated recombinant virus expressing an AIDS virus epitope is placed in a tissue culture flask, such as a recombinant vaccinia virus expressing an AIDS epitope (lXl06-lXl0' plaque-forming units before UV inactivation). /
rnI or AIDS virus epitope related protein or peptide, generally in the range of 1-10 μg/-. After 6-7 days, lymphocytes are stimulated again with the same concentration of recombinant virus, protein or peptide. After 3 days, centrifuge, resuspend in fresh complete medium, and add endotoxin-free recombinant human IL.
-2 (approximately every 1 (11) units of IL-2) is added. Cells are then seeded in a roller bottle at approximately 2×1.0′ cells/rn1 and the roller bottle is continuously rotated at 0.5-1 revolutions per minute.

Cells are about 1X106/all! Cells are seeded again at 2X10' cells/ml when a density of 100 ml is reached. Alternatively, about 1-3X108 cells in complete medium with IL-2 in a hollow fiber cell culture system (e.g. Axis)
st) P [Endotronics ([Endotron
ics, Coon Rapids.

(Minnesota) WA:l, in which the lymphocytes are continuously immersed in fresh medium with [, -2.

Once the total number of cells in the roller bottle or hollow fiber cell culture system reaches approximately 1 x 10I0 cells, it is centrifuged in a conical bottle and the pellet is washed with Hank's Balanced Salt Solution without calcium, magnesium or phenol red. Wash twice. The cells were then rehydrated with 5% normal human serum albumin and 75% normal human serum albumin. (11) 0 units of recombinant human) 0.9% sodium chloride containing IL-2 (11) m
Suspend in infusion medium consisting of 1. The cell suspension is then filtered through a sterile 110 mesh and placed into a Fenwall transfer bag. Samples of cells are tested for the presence of microorganisms, including fungi, aerobic and anaerobic bacteria, and mycoplasma. Samples of cells are retained for immunological testing as described below to demonstrate specific immune transmission.

4.2 Demonstration of specific cell-mediated immunity against AIDS virus-associated epitopes Prior to use in immunotherapy, FJ-enriched lymphocytes are tested for cell-mediated immune reactivity against AIDS virus-infected cells. PBL was identified as an AIDS virus-related epitope (
T by immunofluorescence analysis using monoclonal antibodies directed against T and B cell antigens conjugated with fluorescein after stimulation with recombinant vectors expressing epitope(s) or proteins or peptides containing such epitope(s).
and cell surface expression of B cell markers. An example of such a test is described later in (5,2). Known T cell markers such as CD4 and C
The identity of the lymphocytes activated to express the D8 antigen is confirmed as T cells.

The activated T cells are then tested for reactivity against AIDS virus epitopes.

This could be accomplished by any of several methods known in the art for assaying specific cell-mediated immunity.

For example, a cytotoxicity assay that measures the ability of stimulated T cells to kill a patient's self-AIDS virus-infected cells in vitro can be performed using LAV/HTLVI, a recombinant vaccinia virus expressing AIDS-related epitopes, or a parental vaccinia virus. This can be accomplished by incubating lymphocytes with Cr-labeled cells, such as phytohemdartinin-activated T111 cells, and uninfected labeled cells and measuring ``Cr release upon lysis.'' Such tests have been previously described [e.g. Zarling, J.
, M. et al., 1986, Journal of Immunology (J, Immunol, ) 136: 4
669]. Activated PBLs were also determined by measurement of their proliferative capacity as indicated by 3H-thymidine incorporation after stimulation.
and/or could be tested for T helper cell activity by measuring their ability to produce lymphokines such as IL-2 or interferon when stimulated in the absence of exogenous IL-2. Examples of such assays are described below in specific embodiments of the invention in (5,1), (5,3), (6) and (7). Other known assays for specific cell-mediated immunity, such as the leukocyte adhesion inhibition assay [Tong 7
Thompson, D., M.P.) (ed.)
, 1982, “Assessment of Immune Status by Leukocyte Adhesion Suppression Test”
atus by the Leuko-cyto 8d
herence Inhibition Te5t)
J, Academic Press
ess, New York)] can also be used.

(4,3) Inoculation of Activated T Cells into a Patient The following procedure is a generalized description of the method portion of the invention. Modifications and adaptations of various operations known in the art are within the scope of the invention.

Inoculation of activated T cells is preferably by systemic administration.

T cells can be administered intravenously through a central venous catheter or into a large peripheral vein. Other methods of administration (
For example, direct injection into the artery) is within the scope of the present invention. Approximately 1 x 106 cells are injected initially and the remainder over the next few hours. In some patients, recombinant human
IL-2 can be used and will be infused intravenously every 8 hours starting at the time of T cell infusion. Infusion of IL-2 is preferably carried out at 10.0 as previously used in cancer patients. (1
1) 0-1 (11). (11) The dose would be 0 vehicle position/kg body weight [Rosenberg, S.
A, et al., 1985, New England Journal of Medicine (N, Bngl, J,!
,! ed, ), 313; 1485].

The infusion of IL-2, if tolerated by the patient, will continue for several days after the infusion of activated T cells.

Treatment with inoculation of activated T cells alone or with administration of IL-2 (as described above), interferon, drugs that inhibit AIDS virus replication or monoclonal antibodies against the AIDS virus, or healthy HLA-matched donors. It can be used in conjunction with other therapeutic regimens, including but not limited to the administration of bone marrow transplants from.

Patients can be monitored for evidence of clearance of Lysul antigen-expressing mononuclear lymph in the blood and lymph nodes, pursuing long-term clinical effects on lymph node dysfunction, development of opportunistic infections, Kaposi's sarcoma and other ARC and AIDS-related disorders. can do.

In the following examples, humans exposed to AIDS Lysle or co-pending parent application No. 779.90 referenced herein.
No. 9 (submitted on September 25, 1985); No. 842.984
No. (submitted on March 27, 1986); Agent docket number 56
In vitro activation of T cells using PBL obtained from non-human primates immunized with compositions more fully described in U.S. Application No. 24-026 (filed September 9, 1986). has been proven.

(5) In vitro activation of macaque T cells specific for LAV/HT LV II [In this embodiment of the invention, LAV/1 (TLVI
Cynomolgus monkey 0.1 acacaf immunized with a recombinant virus expressing the envelope glycoprotein II.
LAV/HT in (7 kaku)
In vitro activation of LV-specific T cells is described.

(5,1) Peripheral blood lymphocytes of inoculated macaques are LAV/
Eight feral macaques, which proliferate in response to stimulation with HTLVIII, were treated with L A V / H T L V m
(1) x Envelope glycoproteins tgp41 and hig
Recombinant vaccinia virus (v-
enV5) or vaccinia recombinant virus expressing herpes simplex glycoprotein D-1 (v-HSVgD
Both recombinant viruses were constructed with the wR strain of vaccinia virus. Immunization was carried out by local scarification on the midline of the dorsum using the recombinant virus 2 x 1.
0't Taha 2 x 10' pfu (plaque forming units) was achieved in situ and then a second intradermal immunization was given to macaques 122 weeks later.

Four weeks after the second immunization, peripheral blood lymphocytes (PBL) were isolated from the heparinized blood of immunized macaques by Ficoll-Hypaque centrifugation.

PBL was also used in non-immunized macaques, and v-env5
It was isolated from macaque 81, which had been inoculated only once.

PBL supplemented with 10% heat inactivated normal human serum RP M
I 1640 medium [KIBU: I (GI BCO, Gr.
and Isle, NY). A final volume of 1×10′ PBL in medium was placed in the wells of a round-bottom 96-well plate. For each well, ultraviolet (UV)
0.1ml containing line-inactivated v-env5! Medium (U
V inactivated I
Sucrose gradient centrifugation from supernatant of cellular leukemia system 2
Non-destructive LAV/HTLVIII purified by cycling
'(1 μg/ml, approximately 1 x 10' TCIDs., 50
% tissue culture infectious dose) was added. After 6 days of stimulation, PBL in each well was treated with 1 μC of H-TdR ('H Thymidine, Nev Engla
nd Nuclear.

(Boston, Mass.) for 6 hours, cells were harvested, and incorporated 'H-T'dRcpm was measured by liquid scintillation counting. The results are shown in Table ■. Values shown for uptake of 'H-TdRcem are average values of four replicate wells.Stimulation index indicates the difference between 'H-TdRcpm taken up by stimulated cells and 'H-TdRcem taken up by unstimulated cells. Calculated by dividing.

Table I LAV/HTLV I [[induced proliferative response 67 v-env5 1,586 7.465 4 of 7 PBLs after immunization with LAV/HTLV III envelope glycoprotein expressing vaccinia recombinant virus
．． 7 60.415 3g, 168 v-env5
2,245 9.075 4.0 28,638 12
．． 874 v-env5 1,5B5 4.732
3.0 21.487 13.675 v-env5
5g1 B, 645 14.9 37.847
14.176 v-env5 2.479 5.98
7 2.5 36,657. 14.880 v-e
nv5 1.0? ? 13.922 12.9 24
．． 752 23,081 v-env5 965
3.985 4.1 40.572 42.082
v-env5 2,581 8.580 3.3 46
．． 847 18.173 V-H3VgDl 61
2 553 1.0- 56.217 91.92
6 None 2.911 1.822 1.0-
2.240 1.0-27 None 1.228
1.0?2 1.0- 2.532 2.0 end Incorporated 3H-TdR, cpm 0 copies Stimulation index 8 immunized with v-env5 as shown in Table I
PBLs from all macaques incorporated 2.5-14.9 times more 3H-thymidine than non-stinged IPBLs 6 days after stimulation with LAV/HTLV■. In all of these macaques and in macaque 73 immunized with the V-H3VgD1 recombinant virus, the PBL of v-env5 (Table ■) was higher than that of the unstimulated PBL after stimulation with the parental vaccinia virus.
Incorporated 12.8 to 91.9 times more 3H thymidine. However, non-immunized macaques 26 and 27 or v
- PBL of 73 H3VgD1 immunized macaques was expressed on day 6 after stimulation with various concentrations of LAV/HTLVI [I (Table I
) or LAV/HTL on the 5th or 7th day.
It did not proliferate in response to stimulation with V■. Therefore,
Immunization with v-env5 was performed using LAV/HTLVI[I
specifically induces T cells that proliferate in response to

(5,2) v-env5 or LAV/HTLVII
Peripheral blood lymphocytes activated by I are T cells v-env5 or LAV/HTLVI [To confirm that I-activated PBLs contain T lymphocytes, LAV/HTLVI [I or V PBLs of immunized macaques stimulated with -env5 for 6 days were analyzed by two-color immunofluorescence to detect IL-2 receptors (present on activated T cells and activated B cells), CD4 and CD8 antigens (present on T cells). ), and CD20 (Bp 3
5) Tested for antigen (present on B cells).

This was achieved by the following procedure: PBLs isolated from macaques 47 and 80 after immunization and boosting with recombinant vaccinia virus v-env5 were cultured in medium alone or as previously described (5,1).
) as described in V-env5 or LAV/HT
I was suppressed for 6 days with LVI [[. PBL was then subjected to two-color immunofluorescence using a monoclonal antibody [Clark,
B, A, et al., 1983, Immunocinetics (
Immuno-genetics), 18: 59
9-615] to interleukin receptor (I
L-2R), T cell-associated CD4 and CD8 antigens, and B cell-associated CD20 (Bp35) antigen expression. PBL is a phycoerythrin-conjugated monoclonal antibody 2A3 directed against the interleukin-2 receptor (IL-2R) [Becton Diquintan (Bec.
ton-Dickinsoi+, Inc.

Mountain View, CA)] and CD
Fluorescein-conjugated monoclonal antibody IF against 20
5 [Clark (c1ark, ε, A.) et al., 198
5. Proceedings of the National Academy of Sciences (Proc. Natl.

Acad, Sci,) USA, 82: 1
766-1770) [Cinetic Systems, Inc. (G
enetic Systems Corp,, 5eat
tle, WA), G10-1 against CD8 [
Ledbetter.

J, A, et al., 1985, Journal of I A1
0G (J, Immunol,), 134:4250~
4254) [:Cinetic Systems, Inc. (Gene
tic Systems Carp,, 5eatt
le, WA)] or T4a for CD4 [
Ortho Diagnostics
ostics, Raritan.

(manufactured by NJ)] at the same time. The antibody is modified FAcS■
Sorter [Becton-D
ickinson, Mountain View,
CA) at a saturation concentration determined by titration on lymphocytes analyzed by flow microfluorometry. Quantitative two-color analysis was performed as previously detailed [Ledbetter (
Ledbetter, J. A.) et al., 1984,
“Perspectives in Immunogenetics and Histocompatibility (Perspec)
tivesin Immunogenetics an
d Histocompatibility) J, 6
Volume, Lymphocyte Surface Antigen (Lymphocyte 5ur
face＾nt1gens〔Heidl (E, He1dl
), American Society of Histocompatibility and Immunocinetics (ed.), American Society of Histocompatibility and Immunocinetics
compatibility and [mmunog
enetics, New York),
119-L29 pages]. Forward and right angle scatter gates were set to include lymphoblasts and a substantial number of small lymphocytes. The results are shown in Table ■, and the values shown are for IL-2R+ total percent and CD4, CD8 or CD20(Bl)35) co-expression (
coexpressing) IL-2R'''' cells percentage.

Table■ LAV/HTLVI[[MariIt LAV/H
IL-2 receptors and CD4, CD8 or CD20 (Bp 35) antigens on PBL from inoculated macaques after stimulation with recombinant vaccinia virus (v-env5) expressing T L V m x helobe glycoprotein Macaque PBL IL-2R ” Co-expressed IL
-2R+ cells % cells C[]20 80 V-env5 25.6 62.4 4
0. ONT74 LAv/Hn, vm 14,7
50.0 59.6 1.98OLAV/HTLV
II [12, (158, (138,01,580) No virus OJ 0.3- 0.3- 0.3-The results in Table ■ show that virus infection expressing IL-2 receptor m
Almost all PBLs co-express CD4 or CD8 antigens, but only 1.5-2% co-express CD20 (Bp3
5) Showing co-expression of antigen and indicating that v-env 5 or LAV-HTLVIII activated PBLs are T cells.

(5,3) T cells+ of immunized macaques;! L A V
/) T cells that produce IL-2 after stimulation with ITLV■ or v-env5 produce IL-2-containing lymphokines that can promote differentiation and/or proliferation of T and B cells after antigen stimulation, and Can activate natural killer cells that can lyse cells infected with various viruses including the AIDS virus [Santoli, D.
) et al., 1978, Journal of Immunology, 121:526;
Yasukawa et al. (Yasukawa, M., and 2a
rling.

J9M, ), 1983, Journal of Immunology (J, Immunol, ), 131: 20
11; Ruscetti (F, W,)
et al., 1986, Journal of Immunology)
, 136:3619).

We therefore demonstrated that the T cells of immunized macaques were LAV/'HT.
It was determined whether IL-2 was produced after stimulation with LVIII.

v-e, both constructed with the WP strain of vaccia virus.
PBLs were isolated from heparinized blood of macaques 4 weeks after the second intradermal immunization with nv 5 or v-H5VgD1 (Study 1). V-env 5, v- for test 2
H3VgD1 Matahani x a -y City Sanitation Bureau (Ne
w York C1ty Board of Heal
th) v-en constructed with vaccine strain virus
v5 recombinant virus (v-env5 NY) 2
PBLs were isolated from macaques 4 weeks after primary sarcastic immunization with X 10" pfu. PBLs were suspended in RPM11640 medium supplemented with 10% heat-inactivated normal human blood a, and 2X 10' PBLs were placed in round-bottom 96-well plates. and then UV-inactivated v-env5 (UV
I X 106 pfu/i before radiation inactivation) or
AV/HTLVIII (1 μg/goose) was added to the wells. Supernatants were collected from replicate wells 2 days after stimulation, washed for 24 h before assay, and cultured with IL-2-free IL-2-dependent CTLL-2 cells [Gillis (Or, S, G11)].
lis.

Immunex Carp, 5eattle, W
A) were tested for their ability to support the growth of [provided by].

Cells were labeled with 'H-TdR during the last 6 hours of incubation with supernatant and 38-TdR into cells
Uptake was measured at The units of IL-2 activity present in the supernatant were determined as previously described (Gilli
S, S, et al., 1978, Journal of Immunology (J, Immunol), 120: 2
077), calculated from the standard curve (provided by Or, G11lis) obtained from testing the effects of recombinant and IL-2 on CTLL-2 cell stacks. The results are shown in Table ■.

Table ■ L A V/HT L V m Mata is IL-2 production by PBL of inoculated macaques after stimulation with recombinant vaccinia virus expressing AIDS virus envelope glycoprotein. Kamisumi Mutual No. 1 No Virus LAV/HTLV m v-e
nv5 test 1: 67 v-env5 0 28.0
96.068 v-env5 0
16.0 124.074 v-env
5 0 9.0 52.0?3
v-HSVgDl 0 0 1
0B, 026 None OQ
□Test 2: (13 v-env5 0 14
．． 4 55.8 (15 v-env5NY
0 16.8 33.349 v
-env5NY 0 7.1 26
．． 752, v-env5NY 0
2.4 27.659 v-HSVgDl
0 0 27.626
None OO.

27 None OQ
The results of test 1 in Table Q are for v-env5 or LA from macaques immunized twice with v-env5.
V/HTLVI [Supernatants of stimulated PBL contained IL-2 as demonstrated by their ability to induce proliferation of CTLL-2 cells, an IL-2 dependent cell line. Similarly, as shown in Test 2 in Table ■, IL-2
macaques immunized once with v-enV5 (13) and used as an if vaccine in humans [
Neff, J., 14., 1985, Vaccinia virus as a vector for vaccine antigens.
Vaccinia Viruses as Vecta
rs for Vaccine Antigens)
(Kinan (G, V.

Quinnan, Jr.), Elsevier.

LAV/HTLVI or v from all 37 chickens immunized once with a similar recombinant (v-env 5NY) constructed using the New York City Department of Health strain of vaccinia virus. −
was detected in the supernatant of env5-stimulated PBL. In contrast, PBLs of macaques 73 and 59 immunized with cotton cilia H3VgD1 recombinant virus
produced IL-2 only after stimulation with LAV/HT
After stimulation with LVIII.

This did not occur (Table ■). Unstimulated macaques 26 and 27 did not produce detectable IL-2 after stimulation with LAv/HTLVIII or v-env5. Mainly T cells with helper/inducer activity produce IL-2 after antigen stimulation [Moller.

G, > (4Ji), 1980, Immunol, Rev, vol. 51; iJoretta.

L. et al., 1982, Seminars in Hematol, 19:2?3~
284; Reinherz et al.
L, and Schlossman, S, F,),
1980, Cell, 19:821-827;
Moretsuta (!, Ioretta, ^,), 1985, European Journal of Immunology (Bu
R. J. Immunol.), 15:148-155.
], these results therefore indicate the presence of helper T cells that recognize LAV/HTLVIII in macaques immunized with the recombinant virus.

In addition to their possible role in the differentiation of B cells producing antibodies against LAV/HTLVI [I envelope antigens, these IL-2 producing T cells have cytotoxic T cells capable of killing effector cells such as virus-infected cells. It may be involved in the differentiation and/or expansion of lymphocytes or natural killer cells.

To summarize the results shown in Tables ■, ■, and ■, LAV
Immunization of macaques with a recombinant vaccinia virus expressing the /HTLVIII envelope glycoprotein produces a T cell-mediated immune response against LAV/HTLV! (a) LAV/HTLV! II
proliferation of T cells in response to stimulation by and et al. LAT
I by T cells in response to stimulation with /HTLVIII
It has been shown to generate T cells that can be activated in vitro, as shown by the production of L-2.

The only LAV/HT whose envelope antigen is encoded by the recombinant vaccinia virus used for immunization.
LVII [antigen, so in this particular embodiment LA
It is clear that T cells that proliferate and produce IL-2 after stimulation with V/HTLVI recognize the LAV/HTLV1 envelope antigen (cervical). Envelope antigens and LAV/HTL expressed by recombinant viruses
It is also clear that those expressed by VIII are immunologically cross-reactive.

(6) Example: In vitro activation of chimpanzee T cells specific for LAV/HTLVI
7) PBL is in vitro LAV/HTLVI or LA
Figure 2 shows proliferation in response to stimulation by the V/HTLVIII envelope glycoprotein.

v-env 5NY or herpes simplex virus I
Four weeks after secondary inoculation with a recombinant vaccinia virus (v) ISVgDl) expressing a type glycoprotein, PBLs were separated from hehalinated blood by Ficoll-Hypaque centrifugation. PBL was supplemented with RP supplemented with 10% heat-inactivated normal human serum and penicillin/streptomycin.
96-well plates were seeded at 1×10s cells/well in M11640. Then non-destructive LAV/HTLV
III (5 μg/rrf, purified LAV/HTLVI
LAV/HTLVIII envelope glycoprotein jlt (1 μg/d) isolated by lentil lectin chromatography from I
Before inactivation IXIO' pfu/-), or UV
Inactivated v-env5NY (before UV inactivation I
106 pfu/m were added to replicate wells. After 5 days, 3H thymidine incorporation into the cells was measured by liquid scintillation counting. The results are shown in Table ■.

PBL from all chimpanzees is V-env5NY
Incorporated high levels of 3H-thymidine incorporation after stimulation with (
(based on T cell responses to vaccinia virus). Table■
As shown in , the PBLs of all chimpanzees immunized with V-env5NY were significantly lower than those of v-H3VgD1-immunized chimpanzees (FJα134 and Nα6
4) In contrast to 1: LAV/HTLVVIII and LA
showed a significant proliferative response to the V/HTLVI envelope glycoprotein (env).

(7) Example: In vitro generation of chimpanzee T cell clones with proliferative and cytotoxic activity directed against LAV/HTLVIIIz envelope antigen. We show that T cell clones that do not proliferate and lyse Cr-labeled autologous target cells infected with v-env5, but not the parental vaccinia virus, can be generated in vitro from PBL of chimpanzees immunized with v-env5.PBL Purified AIDS virus envelope antigen, (env.

1 μg/ml) twice at weekly intervals. After 3 days, the stimulated cells were cloned into the wells of a 96-well plate by limiting dilution and supplemented with 10% depleted IL-2 (
CellularProdu7
cts, Buffalo, NY) in complete medium containing 5×10′ X-irradiated (250 OR) autologous PBL (and UV inactivated). Clone V-env5
and expanded by late feeding with X-irradiated PBL in IL-2 containing medium. To test for proliferative responses to V-env5 and vaccinia virus, cloned T cells were washed to remove IL-2.
lXl0' cloned T cells were incubated with 5X10' X-irradiated autologous PBL and v-e in IL-2-free medium.
nv5 or vaccinia virus (before UV inactivation 1
×10” pfu/ml) in replicate wells.

After 3 days, 3H thymidine was added to the wells, and 3H thymidine incorporated during 6 hours was measured by liquid scintillation counting. The results in Table V show that all four clones proliferate in response to v-env5, but not the parental vaccinia virus.

Cloned cells were also infected with v-env5, vaccinia virus, or not infected with the virus! +(r
Their ability to lyse labeled autologous lymphoblastoid target cells was tested in a 6-hour %+Cr release assay using a cloned effector cell:target cell ratio of 25:1. The results are shown in Table V. Cloned cells are v-env
5 infected cells, but not parental vaccinia infected or uninfected cells, thus demonstrating their specificity for the AIDS virus envelope antigen (cervical).

Table V Proliferation and Cytotoxic Activity of Cloned T Cells from v-env5 Immunized Chimpanzee #124 1 5.31? 33.907 8,2
722 4.997 15,225 6,
3423 1.090 9.080 2.
91 (14 4.607 13, (150
5,392L -2,420,2-0,12
-0,77,0-1,3 3-0,011,01,0 (8) Example: In vitro activation of human T cells by v-env5 The following test was performed on PBL of AIDS virus seropositive individuals. This shows that the cells proliferate in response to stimulation by v-env5 within the tube. PBLs were separated by Ficoll-Hyvac centrifugation and resuspended in RPM11640 medium containing 10% heat-inactivated pooled normal human serum. 0.1 ml PBL (IXIO' cells) was then added to each of 4 replicate wells, followed by 0.1-medium (no antigen), 0.
．． 1-ultraviolet (UV) inactivationv-env5 (UV
Allogeneic PBL (108 pfu/mf before inactivation) or 1 x 10% X-ray irradiation (250 OR') was added. 3 after 6 days
H-thymidine (3H7dR) was added to the wells (0.5 μC
i/well) and 6 hours later, cells were harvested using a multiwell harvester. 3H-TdR uptake was measured by liquid scintillation counting. The stimulation index (SI) is determined by dividing the ffH-TdR cpm taken up into stimulated cells by the cpm taken up into unstimulated cells.
was calculated. The results are shown in Table ■.

As can be seen from Table V, PBL from both seropositive and seronegative donors showed proliferation in response to v-env5. PBL generally from seropositive donors
showed a lower response than seronegative donors. but,
PBLs from seropositive donors show a significant response and activated T cells can expand in IL-2 containing cell culture.

(9) Deposit of microorganisms The following recombinant viruses are stored in the American Type Culture Collection (American Type Culture Collection).
1ture Collection, Rockville
e, MO) and designated the indicated accession number: Recombinant virus accession number v-env2A
TCCVR2114 v -env5 ATCCVR2113v
-env7 ATCCVR21
48v-env5NY ATCCVR214
9v-gag1NY ATCCVR2150
Ac-g, agl ATCC VR2147A
The c-env5 ATCC VR2151 deposited embodiment is intended as merely illustrative of one aspect of the invention; the invention extends beyond the deposited recombinant virus as any functionally equivalent recombinant can be used within the scope of the invention. It should not be limited.

Various modifications of the invention in addition to those actually shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the claims.

Claims (40)

[Claims] (1) A method of immunotherapy for treating a patient infected with the AIDS virus, comprising: (a) the activated T lymphocytes are specific for an epitope of the AIDS virus and are histocompatible with the patient; b) Activated T lymphocytes are incubated with radiolabeled target cells from a patient infected with the AIDS virus, and the release of the radiolabel causes lysis of infected target cells. (ii) a stimulation assay that measures the ability of activated T lymphocytes to proliferate and/or produce lymphokines in response to stimulation with AIDS virus epitopes in vitro; administering to the patient activated T lymphocytes capable of mediating AIDS virus-specific T cell immunity, as measured by any Method. (2) The method according to claim (1), wherein the activated T lymphocytes are activated in the test by exposure to an epitope of the AIDS virus. (3) The method according to claim (2), wherein the epitope of the AIDS virus includes an envelope epitope. (4) The method according to claim (2), wherein the epitope of the AIDS virus includes a gag epitope. (5) The method according to claim (1), wherein the activated T lymphocytes are activated in vitro by exposure to a peptide associated with an epitope of the AIDS virus. (6) The method according to claim (5), wherein the epitope of the AIDS virus includes an envelope epitope. (7) The method according to claim (5), wherein the epitope of the AIDS virus includes a gag epitope. (8) The method according to claim (1), wherein the activated T lymphocytes are activated in vitro by exposure to a recombinant microorganism expressing an AIDS virus epitope. (9) The method according to claim (8), wherein the AIDS virus epitope includes an envelope epitope. (10) The method according to claim (8), wherein the AIDS virus epitope includes a gag epitope. (11) Claims (8), (9), or (10), wherein the recombinant microorganism includes a recombinant virus.
The method described in section. (12) The method according to claim (11), wherein the recombinant virus includes an animal virus. (13) The method according to claim (12), wherein the animal virus includes vaccinia virus. (14) The method according to claim (11), wherein the recombinant virus includes an insect virus. (15) The method according to claim (14), wherein the insect virus includes baculovirus. (16) Activated T lymphocytes were deposited with ATCC and assigned accession number ATCC VR2113.
5. The method of claim (1), activated by exposure to v5. (17) Activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2149.
The method of claim (1), wherein the method is activated by exposure to v5NY. (18) Activated T lymphocytes are v-ga deposited with ATCC and designated accession number ATCC VR2150.
The method of claim (1), activated by exposure to g1NY. (19) Activated T lymphocytes are Ac-e deposited with ATCC and assigned accession number ATCC VR2151.
A method according to claim 1, wherein the method is activated by exposure to nv5. (20) Ac-g activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2147.
The method of claim (1), wherein the method is activated by exposure to ag1. (21) A method of immunotherapeutically treating a patient infected with the AIDS virus, comprising the steps of: (a) isolating lymphocytes from the patient or a histocompatible blood donor exposed to AIDS virus epitopes; (c) The following assay method: (i) Expose the activated T lymphocytes to AIDS virus epitopes in vitro to activate T lymphocytes specific for the AIDS virus. (ii) cytotoxicity assays in which cells are incubated with radiolabeled target cells from infected patients and the release of radiolabel is indicative of lysis of the infected target cells, and (ii) proliferation and proliferation in vitro in response to stimulation with AIDS virus epitopes. (or) a stimulation assay that measures the ability of activated T lymphocytes to produce lymphokines; the activated T lymphocytes at stage (b) capable of mediating AIDS virus-specific T cell immunity as measured by: (d) injecting into the patient an effective amount of the activated T lymphocytes of step (c) to transfer T cell-mediated immunity directed against cells infected with the AIDS virus. (22) The method according to claim (21), wherein the activated T lymphocytes are activated in vitro by exposure to an epitope of the AIDS virus. (23) The method according to claim (22), wherein the AIDS virus epitope includes an envelope epitope. (24) The method according to claim (22), wherein the AIDS virus epitope includes a gag epitope. (25) The method according to claim (21), wherein the activated T lymphocytes are activated in vitro by exposure to a peptide related to an epitope of the AIDS virus. (26) The method according to claim (25), wherein the epitope of the AIDS virus includes an envelope epitope. (27) The method according to claim (25), wherein the epitope of the AIDS virus includes a gag epitope. (28) The method according to claim (21), wherein the activated T lymphocytes are activated in vitro by exposure to a recombinant microorganism expressing an AIDS virus epitope. (29) The method according to claim (28), wherein the AIDS virus epitope includes an envelope epitope. (30) The method according to claim (28), wherein the AIDS virus epitope includes a gag epitope. (31) Claims (28), (29), or (3), wherein the recombinant microorganism includes a recombinant virus.
0) The method described in section 0). (32) The method according to claim (31), wherein the recombinant virus includes an animal virus. (33) The method according to claim (32), wherein the animal virus includes vaccinia virus. (34) The method according to claim (31), wherein the recombinant virus comprises an insect virus. (35) The method according to claim (34), wherein the insect virus includes baculovirus. (36) Activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2113.
22. The method of claim 21, wherein the method is activated by exposure to v5. (37) Activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2149.
22. The method of claim 21, wherein the method is activated by exposure to v5NY. (38) Activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2150.
22. The method of claim 21, wherein the method is activated by exposure to g1NY. (39) Activated T lymphocytes were deposited with the ATCC and assigned accession number ATCC VR2151.
22. The method of claim 21, wherein the method is activated by exposure to nv5. (40) Ac-g activated T lymphocytes were deposited with ATCC and designated accession number ATCC VR2147.
The method according to claim 21, wherein the method is activated by exposure to ag1.